Electronic measurements of monolayers following homogenous reactions of their components

Abstract

The disclosure relates to novel methods for performing a solution based assay reaction with an electroactive active moiety (EAM) that subsequently forms a self-assembled monolayer (SAM) utilizing the advantages of faster solution reaction kinetics, SAM protected electrode and surface based electrochemistry for electronic measurement.

1 . A method for detecting a target analyte in a test sample, said method comprising:
 (a) contacting a test sample with a capture binding ligand that binds to a target analyte, under conditions wherein said capture binding ligand binds said target analyte, if present, in said test sample to form a first complex, and wherein said capture binding ligand bound to a first solid support;   (b) contacting said first complex with a second binding ligand under conditions wherein said first complex and said second binding ligand bind to form a second complex, wherein said second binding ligand is indirectly bound to an intermediary enzyme of a peroxide-generating system;   (c) isolating said second complex;   (d) contacting said second complex with a substrate for said intermediary enzyme of peroxide-generating system under conditions such that products are generated to form a first assay mixture;   (e) contacting a peroxide-generating enzyme with said first assay mixture under conditions wherein peroxide is generated to form a peroxide-containing second assay mixture;   (f) contacting said peroxide containing second assay mixture with an electroactive moiety (EAM) comprising a transition metal complex, a self-immolative moiety (SIM), and a peroxide sensitive moiety (PSM), wherein said SIM joins the PSM to the transition metal complex and wherein said EAM has a first E 0  when said SIM and PSM are present, to form a third assay mixture wherein said peroxide reacts in the solution phase with said PSM of said EAM to release said SIM from said EAM and result in said EAM having a second E 0 ;   (g) contacting said third assay mixture with a second solid support comprising an electrode under conditions such that a covalently attached self-assembled monolayer (SAM) forms comprising said EAM with said first E 0  and with said second E 0 ; and   (h) detecting for a change between the first E 0  and the second E 0  of said EAM, wherein said change is an indication of the presence of said target analyte.   
     
     
         2 - 15 . (canceled) 
     
     
         16 . The method of  claim 1 , wherein said indirect binding between said second binding ligand and said intermediary enyzme is carried out as a separate step and wherein step (b) further comprising:
 (bi) contacting said first complex with said second binding ligand under conditions wherein said first complex and said second binding ligand bind to form a second complex; and   (bii) contacting said second complex with said intermediary enzyme of said peroxide-generating system under conditions wherein said intermediary enzyme becomes indirectly bound to said second complex.   
     
     
         17 . The method of  claim 1 , wherein said intermediary enzyme is indirectly bound to said second binding ligand through a biotin-streptavidin interaction. 
     
     
         18 . The method of claim  2 , wherein said intermediary enzyme is indirectly bound to said second binding ligand through a biotin-streptavidin interaction. 
     
     
         19 . The method of  claim 1 , wherein the target analyte is a protein. 
     
     
         20 . The method of  claim 1 , wherein said first solid support is selected from the group consisting of microparticles, magnetic microparticles, beads, and microchannels. 
     
     
         21 . The method of  claim 1 , wherein said products are a substrate for a peroxide-generating enzyme. 
     
     
         22 . The method of  claim 1 , further comprising the presence of a substrate for said peroxide-generating enzyme and wherein said products are a cofactor for said peroxide-generating enzyme. 
     
     
         23 . The method of  claim 1 , wherein said intermediary enzyme of a peroxide-generating system is a dephosphorylating enzyme. 
     
     
         24 . The method of  claim 23 , wherein said dephosphorylating enzyme is alkaline phosphatase. 
     
     
         25 . The method of  claim 1 , wherein said peroxide-generating enzyme is a flavin dependent oxidoreductase enzyme. 
     
     
         26 . The method of  claim 25 , wherein said flavin dependent oxidoreductase enzyme is D-amino acid oxidase. 
     
     
         27 . The method of  claim 1 , wherein said peroxide-generating enzyme of a peroxide-generating system is an oxidase enzyme. 
     
     
         28 . The method of  claim 27 , wherein said peroxide-generating enzyme is glucose oxidase. 
     
     
         29 . The method of  claim 1 , wherein said first binding ligand and said second binding ligand are independently selected from the group consisting of monoclonal antibodies, fragments of monoclonal antibodies, polyclonal antibodies, fragments of polyclonal antibodies, proteins, and peptides. 
     
     
         30 . The method of  claim 1 , wherein said transition metal complex comprises iron, ruthenium, or osmium. 
     
     
         31 . The method of  claim 1 , wherein said transition metal complex is a ferrocene or substituted ferrocene.